WO1999061128A1 - Systems for column-based separations, methods of forming packed columns, and methods of purifying sample components - Google Patents
Systems for column-based separations, methods of forming packed columns, and methods of purifying sample components Download PDFInfo
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- WO1999061128A1 WO1999061128A1 PCT/US1999/011829 US9911829W WO9961128A1 WO 1999061128 A1 WO1999061128 A1 WO 1999061128A1 US 9911829 W US9911829 W US 9911829W WO 9961128 A1 WO9961128 A1 WO 9961128A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/58—Conditioning of the sorbent material or stationary liquid the sorbent moving as a whole
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
- B01D15/206—Packing or coating
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/56—Packing methods or coating methods
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/56—Packing methods or coating methods
- G01N2030/562—Packing methods or coating methods packing
- G01N2030/565—Packing methods or coating methods packing slurry packing
Definitions
- the invention pertains to systems for column-based separation, and to methods of forming and utilizing packed columns. In specific embodiments, the invention pertains to methods of separating sample components.
- Column-based separations are frequently used for selectively removing components from mixtures.
- a first step in utilizing column-based technology is to form a column. Such can be accomplished within a column chamber.
- An exemplary prior art column chamber 10 is illustrated in Fig. 1.
- Column chamber 10 comprises a longitudinal tubular section 12 having ends 14 and 16.
- An inlet 18 is provided at end 16, and an outlet 20 is provided at end 14.
- Outlet 20 is obstructed by a porous filter 22.
- Filter 22 can comprise, for example, a porous fritted glass or ceramic material.
- a packed column is formed within chamber 10 by flowing a slurry comprising a mixture of matrix material 15 and carrier fluid 17 into inlet 18.
- Matrix material 15 comprises a plurality of particulates, such as, for example, beads.
- Filter 22 is permeable to the carrier fluid and impermeable to the matrix material. Accordingly, as the slurry is flowed into column chamber 10, matrix material 15 stacks against filter 22 to form a packed column 19 within tubular portion 12.
- the composition of carrier fluid 17 and matrix material 15 vary depending on the components that are intended to be separated by the packed column, and on the mixtures (samples) within which such components are found.
- Example matrix materials are Sr-resin, TRU-resin, and TEVA-resin, all of which can be obtained from ElChrom Industries, Inc., of Darien, Illinois. Such matrix materials can have particle sizes in the range of, for example, 20-100 micrometers.
- Sr- resin, TRU-resin, and TEVA-resin can be used for, for example, selectively retaining radioactive materials. Specifically, Sr-resin can selectively retain strontium, TRU-resin can selectively retain americium, and TEVA-resin can selectively retain technetium.
- Slurries utilized for forming packed columns of Sr- resin, TEVA-resin, or TRU-resin can comprise, for example, 0.074 gram/mL of Sr-resin in 3 M HNO 3 ; 0.142 grams/mL of TEVA-resin in 4 M HNO 3 . or 0.076 grams/mL of TRU-resin in 0.1 M HNO 3 , respectively.
- nucleic acid is defined to include DNA nucleotides and RNA nucleotides, as well as any length polymer comprising DNA nucleotides or RNA nucleotides.
- the column-based separations generally have in common that a mixture in a first physical state (typically either a gas phase or a liquid phase) is flowed across a column matrix in a second physical state (typically either a liquid phase or a solid phase) to separate a component of the mixture from other materials of the mixture. Accordingly, the physical state of the matrix is generally different than the physical state of the component that is to be separated.
- a method of extracting a retained component is to subject the column matrix to conditions which disrupt interactions between the matrix material and the component to thereby elute the component from the matrix material. In some applications, it is desirable to elute the retained material from the matrix material while the matrix material is still within a packed column, and in other applications it is desirable to remove the matrix material from a packed column before eluting the retained component.
- a difficulty in utilizing column-based separations is in removing matrix material from a column chamber and subsequently repacking additional matrix material in the chamber to re-form a packed column.
- a matrix material of a packed column can be rendered unusable after an initial separation, or after an initial series of separations.
- a matrix material can be rendered unusable if it is degraded by fluids passed through the material during a separation.
- the matrix material can be rendered unusable if it becomes contaminated by materials within a sample because such contamination can pose a risk of cross- contamination.
- apparatus 30 comprises a tubular column chamber 32 having an inlet end 34 and an outlet end 36. Outlet end 36 terminates proximate a plate 38. Plate 38 can comprise a window configured to enable light to pass through for spectroscopic measurement of materials eluting from column chamber 30.
- a matrix material 40 forms a packed column 42 within column chamber 32.
- Packed column 42 has a lateral periphery defined by tubular chamber 32.
- Packed column 42 can be formed by flowing a slurry of matrix material 40 and a carrier fluid into column chamber 32.
- Outlet end 36 of column chamber 32 is displaced from plate 38 by a distance "D" sufficient to enable the carrier fluid to pass between column chamber 32 and plate 38. However, the distance is less than an average width of matrix material 40. Accordingly, matrix material 40 is retained in column chamber 32 and stacks against plate 38 to form packed column 42.
- Fig. 3 illustrates a method for removal of matrix material 40 from packed column 42. Specifically, column chamber 32 is raised to enable matrix material
- System 30 is improved relative to other methods of packing and unpacking columns in that it can provide a quick method for releasing packed column w material from a column chamber, and can also provide a quick method for resetting the column chamber to be repacked with fresh matrix material.
- a difficulty with column system 30 is that it can be problematic to move an entirety of column chamber 32 during transitions between packing and unpacking operations. Further, precise tolerances are needed to hold beads and may leak
- Discharged beads can undesirably pass through a detector. It can become increasingly difficult to move the entirety of column chamber 32 as a column- based separation is scaled up for larger operations. Accordingly, it is desirable to develop alternative methods for conveniently packing and unpacking column chambers, wherein a column chamber is not moved in transitioning between
- the invention encompasses a method of packing and unpacking a column chamber.
- a matrix material is packed within a column chamber to form a packed column. After the packing, the matrix material is
- the invention encompasses a method of purifying a component of a sample.
- a column chamber having an inlet end and an outlet end is provided.
- the outlet end terminates proximate both a first flow path and a second flow path.
- the first flow path is obstructed with a retaining material
- the second flow path is blocked by a blocking material that removably blocks flow of both the first fluid and the column matrix material.
- a suspension of the first fluid and the matrix material is flowed into the column chamber and along the first flow path to form a packed column of the matrix material within the column chamber.
- the blocking material defines a portion of a periphery of the packed column.
- the matrix material is configured to selectively retain a component of the sample. The sample is flowed through the packed column and along the first flow path to separate the component from the rest of the sample. The blocking material is removed without moving the column chamber. After removing the blocking material, a second fluid is flowed through the column chamber and along the second flow path to remove the matrix material from the column chamber.
- the invention encompasses a system for column-based separations.
- the system comprises a fluid passageway containing a column chamber and a flow path in fluid communication with the column chamber.
- the flow path is obstructed by a retaining material permeable to a carrier fluid and impermeable to a column matrix material suspended in the carrier fluid.
- the flow path extends through the column chamber and through the retaining material.
- the flow path is configured to form a packed column within the column chamber when a suspension of the fluid and the column matrix material is flowed along the flow path.
- the column chamber defines a portion of a periphery configured to retain the packed column. Another portion of the periphery is defined by a blocking material that removably blocks flow of both the carrier fluid and the column matrix material. The blocking material is spaced from the packed column by a region configured to retain a fluid.
- An advantage of the impermeable material is that the surface area of the material in contact with fluid is always in contact with fluid. In other words, there is no material surface area that alternately contacts fluid then, say an interior chamber surface. This feature minimizes the potential of sample to sample contamination since a sample may be completely washed through and not captured on an alternately or intermittently exposed surface. This is especially valuable for nucleic acid samples wherein one molecule of a previous nucleic acid sample can be detected in a subsequent nucleic acid sample.
- Fig. 1 is a diagrammatic, cross-sectional view of a prior art column construction.
- Fig. 2 is a diagrammatic, cross-sectional view of a prior art system for packing and unpacking a column chamber.
- the system of Fig. 2 is shown with the column chamber in a position for packing a matrix material within the column chamber.
- Fig. 3 is a view of the Fig. 2 system, with the column chamber shown in a position for unpacking the column chamber.
- Fig. 4 is a diagrammatic, cross-sectional view of a first embodiment system of the present invention for packing and unpacking a column chamber.
- the system of Fig. 4 is shown in a position for packing a column chamber.
- Fig. 5 is a view of the Fig. 4 system shown in a position for unpacking the column chamber.
- Fig. 6 is a view of a second embodiment system of the present invention for packing and unpacking a column chamber.
- Fig. 6 is a view of the system in a position for packing the column chamber.
- Fig. 7 is a view of the Fig. 6 system shown in a position for unpacking a column chamber.
- Fig. 8 is a schematic view of a first embodiment sample treatment apparatus of the present invention.
- the apparatus of Fig. 8 incorporates a third embodiment packing and unpacking system.
- Fig. 9 is a schematic view of a second embodiment sample treatment apparatus of the present invention.
- Fig. 10 is a schematic view of another embodiment of the present invention. Best Modes for Carrying Out the Invention and Disclosure of Invention
- the invention encompasses systems for column-based separations configured to pack and unpack column chambers without moving the column chambers.
- Embodiments pertaining to this aspect of the invention are described with reference to Figs. 4-8, with Figs. 4 and 5 illustrating a first embodiment, Figs. 6 and 7 illustrating a second embodiment, and Fig. 8 illustrating a third embodiment.
- a system 50 for column-based separations comprises a column chamber 52 having an inlet end 54 and an outlet end 56.
- Column chamber 52 comprises a longitudinal axis "X", and can be formed of, for example, glass, plastic or metal.
- column chamber 52 comprises a pair of opposing sidewalls 53 and 55.
- sidewalls 53 and 55 are shown as physically separated in the shown cross- sectional view, it is to be understood that sidewalls 53 and 55 can be portions of a continuous periphery.
- column chamber 52 can have a cylindrical shape, with sidewalls 53 and 55 forming portions of a continuous circular periphery of the cylinder.
- Outlet end 56 is obstructed with a retaining material 58 which is permeable to a carrier fluid and impermeable to a column matrix material suspended in the carrier fluid.
- Retaining material 58 can comprise, for example, a porous fritted glass and/or ceramic material.
- a first flow path for fluids passing through column chamber 52 is defined to extend through retaining material 58.
- a first valve 64 is provided to selectively block flow along the first flow path. First valve 64 is shown in a "open" position in Fig. 4, enabling a fluid to flow along the first flow path.
- System 50 further comprises a tube 60, which in the shown preferred embodiment extends to the longitudinal axis "X" of column chamber 52. It is further preferred that tube 60 extend at about a right angle to the longitudinal axis "X”.
- a rotatable second valve 62 is provided within tube 60 to regulate flow of material through tube 60. Second valve 62 is configured to block flow of both fluids and matrix material.
- Fig. 4 illustrates system 50 with second valve 62 in a "closed” position to prevent flow of fluid and matrix material through tube 60.
- Tube 60 defines a second flow path for materials flowed into column chamber 52, and in Fig. 4 such second flow path is illustrated as being blocked by closed valve 62.
- sidewall 53 terminates in a fluid tight seal at retaining material 58
- sidewall 55 terminates at a location elevationally displaced from retaining material 58.
- Second valve 62 is laterally displaced from column chamber 52 and can be used to regulate flow of materials under sidewall 55. Specifically, when valve 62 is in a closed position, flow under sidewall 55 is prevented, and when valve 62 is in an open position, flow under sidewall 55 is enabled.
- a support structure 66 is provided to support column chamber 52 and tube 60.
- Support structure 66 can comprise, for example, a plastic material molded to fit column chamber 52 and tube 60.
- support structure 60 can comprise, for example, a clamp.
- a matrix material 68 is provided within column chamber 52. In the Fig.
- closed valve 62 defines second portion 74 through a retaining fluid 76 that is within a fluid-filled space extending between valve 62 and packed column 70.
- Retaining fluid 76 forms a boundary along which matrix material 68 is packed to form packed column 70.
- retaining fluid 76 forms a barrier which precludes matrix material 68 from entering the fluid-filled space between valve 62 and packed column 70 during formation of packed column 70.
- the retaining fluid 76 can be either a gas or a liquid, but is preferably a liquid.
- the carrier fluid is preferably a liquid.
- tube 60 and column chamber 52 are initially filled with a first liquid prior to introduction of a slurry comprising a liquid carrier fluid and a matrix material within column chamber 52.
- the liquid carrier fluid then flows through outlet 56, while the first liquid remains within tube 60 to form a retaining fluid volume between valve 62 and packed column 70.
- a pressure may be exerted on retaining fluid 76 to elevate a pressure of retaining fluid 76 above one atmosphere. This can occur, for example, if pressure is utilized to force carrier fluid through retaining material 58.
- valve 64 in a closed position and valve 62 in an open position. Opening valve 62 unblocks flow of matrix material along tube 60, enabling discharge of matrix material 68 through tube 60.
- a fluid preferably a liquid
- the fluid flowed during discharge of matrix 68 can be referred to as a dislodging fluid, and can be the same as one or both of the carrier fluid and the retaining fluid 76 (Fig. 4).
- the system 50 described with reference to Figs. 4 and 5 can be shifted from a packing mode to an unpacking mode by activating two valves. Specifically, when valve 62 is in a closed position and valve 64 is in an open position, system 50 is in a column chamber packing mode (Fig. 4), and when valve 62 is in an open position and valve 64 is in a closed position, system 50 is in a column chamber unpacking mode (Fig. 5).
- Figs. 4 and 5 enables a system to be shifted from a column chamber packing mode to a column chamber unpacking mode without moving the column chamber.
- Fig. 6 illustrates column system 80 in a column chamber packing mode
- Fig. 7 illustrates column system 80 in a column chamber unpacking mode
- column system 80 comprises a column chamber 82 having an inlet end 84 and an outlet end 86. Outlet end 86 is obstructed with a retaining material 88 permeable to a carrier fluid, and impermeable to a matrix material. Retaining material 88 can comprise, for example, a porous glass or ceramic material.
- System 80 further comprises a valve 102 having at least four ports and two positions. The four ports are labeled as 104, 106, 108, and 110.
- Port 110 is blocked with a blocking material 90.
- Ports 104 and 108 are in fluid communication with column chamber 82, and comprise a first flow path and a second flow path, respectively, from column chamber 82.
- Port 106 is an unblocked outlet port which can lead to, for example, a detector, a collection chamber, or additional equipment for processing substances eluted from packed column 92.
- Blocking material 90 is configured to block flow of both column matrix material and carrier fluid, and can comprise, for example, a plastic plug threadably inserted into port 110, or a suitable valve.
- a packed column 92 of matrix material 94 is formed within column chamber 80.
- Column chamber 80 defines a portion 96 of a periphery of packed column 92, and blocking material 90 defines another portion 98 of the periphery of packed column 92.
- Blocking material 90 is separated from the periphery of packed column 92 by a fluid-filled region 100 filled with a retaining fluid 101.
- Fluid-filled region 100 can comprise, for example, a plastic tubing.
- column chamber 82 comprises a right-angle bend 112 proximate retaining material 88, and accordingly proximate an outlet extending through material 88.
- the portion 98 of the periphery of packed column 92 defined by the retaining fluid 101 is at right-angle bend 112.
- An alternative method of describing the apparatus of system 80 is to describe column chamber 82 as comprising a longitudinal section terminating at right-angle bend 1 12, and further comprising a fluid filled region 100 comprising a tube at right angle bend 1 12.
- the longitudinal section of column chamber 82, together with right-angle bend 1 12, and the tubular portion of fluid filled region 100 that joins with right-angle 1 12 define a "T" shape in a zone illustrated as zone "Z". Referring to Fig.
- valve 102 moved into a second position which couples port 104 with blocked port 110 and which couples port 108 with open port 106.
- the second position of valve 102 thus blocks the first flow path through retaining material 88 and opens the second flow path adjacent column chamber 82 to enable discharge of matrix material 94 from column chamber 82.
- Figs. 4-7 can enable a column chamber to be packed and unpacked without moving the column chamber.
- the embodiments utilize a blocking material (either valve 62 or material 90) to removably block flow of fluids and column matrix material from a column chamber. When flow is blocked, the column chamber can be packed with a matrix material, and when the flow is unblocked the column chamber can be unpacked.
- the column chambers of Figs 4-7 are preferably packed and unpacked with matrix material while flowing fluids through the column chambers in an identical flow direction during both the packing and unpacking operations. Specifically, all, or at least most, of the matrix material is preferably expelled from a column chamber along an identical flow direction as was utilized to pack the column chamber.
- the invention also encompasses embodiments in which flow of fluid through a column chamber is reversed during a packing or unpacking operation.
- the flow during a packing operation will not be reversed, but will instead be continuous in a flow direction from an inlet of the column chamber through a retaining material (such as retaining material 88 of Figs. 6 and 7).
- a flow of fluid through a column chamber will be predominately in a single direction from an inlet of the column through an outlet of the column during an unpacking operation.
- the unpacking operation can preferably also comprise some sporadic instances wherein flow is reversed (i.e., to go from an outlet to an inlet) to assist in dislodging matrix material from within column chamber 82, and to assist in removing matrix material embedded within a porous retaining material (such as retaining material 88 of Figs. 6 and 7) that could otherwise potentially clog the retaining material.
- Embodiments of the present invention can be operated with a vast number of matrix materials, as will be appreciated by persons of ordinary skill in the art.
- Example matrix materials are the Sr-resin, TRU-resin, and TEVA-resin described above in the "Background" section of this disclosure, which can be utilized for separating compounds comprising radioactive atoms from other materials in a sample.
- Additional example matrix materials including but not limited to glass, sepharose, polystyrene, Tepnel, Qiagen, zirconium, hydroxyapatite, POROS, PEG- PS, and PS the last three of which are made by PerSeptive are materials suitable for separating biological materials.
- Certain matrix materials are materials for separating nucleotide fragments (e.g., nucleic acid, DNA, RNA or combinations thereof) based upon a sequence of the fragments, such as, for example, the Tepnel micro-beads discussed above in the "Background" section of this disclosure.
- Biological materials include but are not limited to viruses, cells for example prokaryote, eukaryote and combinations thereof.
- Column systems of the present invention can be incorporated into methods for purifying components of samples.
- Example purification apparatuses are shown in Figs. 8 and 9, with Fig. 8 showing a first embodiment apparatus and Fig. 9 showing a second embodiment apparatus.
- a purification apparatus 120 comprises a pump 122 (for example, a syringe pump), valves 124, 126, 128 and 130, and a column chamber 132.
- Exemplary valves 128 and 130 are 4 port, 2 position diverter valves such as may be obtained from, for example, Valco as Cheminert valves.
- Valve 130 comprises ports 140, 142, 144 and 146, with port 146 obstructed by a filter 148.
- Filter 148 can comprise, for example, a 25 ⁇ m poresize polypropylene column end frit with a 5 mm diameter, and can be placed into a port of a 2 position diverter valve with a flat-bottom chromatographic fitting.
- Column chamber 132 is packed by flowing a slurry of carrier fluid and matrix material into column chamber 132 while a fluid flow is coupled through ports 144 and 146. Filter 148 is permeable to the carrier fluid and impermeable to the matrix material. Column chamber 132 can be subsequently unpacked by activating valve 130 to couple a fluid flow through ports 144 and 142. Port 142 is preferably an open port sized to permit flow of a slurry comprising matrix material through port 142. Column chamber 132 and valve 130 together comprise a third embodiment column system of the present invention that can be packed and unpacked without moving a column chamber.
- Valve 128 comprises ports 150, 152, 154 and 156.
- Port 152 can lead to, for example, a waste reservoir (labeled "W").
- Port 150 is preferably in fluid communication with a wide dimension tubing 151, such as, for example, tubing having an internal diameter of 1.6 mm, to enable a slurry comprising matrix material and carrier solution to easily flow through the wide bore tubing during packing of column 32.
- Port 154 is preferably in fluid communication with tubing 155 having a relatively narrow bore, such as, for example, an internal diameter of 0.8 mm. Such narrow bore tubing enables a sample to be flowed onto column 32 with less dilution of the sample than would occur with a wider diameter tubing.
- a fluid flow is coupled through ports 150 and 156 during packing of column 32, and a fluid flow is coupled through ports 154 and 156 during loading of a sample onto column 152.
- Either of ports 154 or 150 can be coupled to port 156 during elution of sample from matrix material of column 132.
- a sample eluted from the matrix material of column 132 can be passed through filter 148 to a detector 170 downstream of port 146.
- Detector 170 can comprise, for example, a polymerase chain reaction (PCR) machine.
- Valves 126 and 124 are utilized for providing reagents, samples, and slurries to column 132.
- tubing segment 172 that can be any shape.
- Tubing segment 172 is preferably coiled as shown to save space.
- the slurry subsequently flow from tubing segment 172 to column 132.
- the slurry can be provided within apparatus 120 without introduction of gas bubbles into the tubing of the apparatus.
- tubing segment 172 can be filled with a liquid (preferably a liquid inert to reaction with the slurry) prior to injection of slurry into holding coil 172.
- the slurry can then be injected into tubing segment 172 to displace the liquid.
- a small air bubble (about 100 microliters) can be introduced into tubing segment 172 prior to injecting the slurry.
- Each of valves 126, 128 and 130 preferably comprises a port going to a waste material reservoir "W".
- Example dimensions of various items of apparatus 120 are as follows: column 132 can comprise a volume of about 250 ⁇ L, holding coil 172 can comprise a void volume of about 12 mL, and syringe pump 122 can comprise a displacement of about 10 mL.
- a typical time for packing a 250 microliter column 132 is less than four and one-half minutes.
- the holding coil, column chamber and tubing of apparatus 120 can comprise, for example, TEFLONTM, with the holding coil and tubing preferably comprising FEP(fluorinated ethylene propylene)-TEFLON and the column chamber preferably comprising PTFE(polytetrafluoroethylene)-TEFLON.
- Example conditions for utilizing apparatus 120 are described in Tables 1-5.
- steps 3 and 4 were executed 3 times to pack Sr-resin and TRU-resin columns, and 2 times to pack TEVA-resin column.
- Table 2 Automated Protocol for Sorbent Bed Disposal (Described With Reference To The Apparatus Of Fig. 8)
- the sample zone in step 3 is sequenced between 8M HNO 3 -0.12M HF eluent zones; aspiration sequence: 6mL eluent 8M HNO 3 -0.12M HF/100 ⁇ L sample/ 150 ⁇ L eluent.
- the flow rate in step #3 was 1 mL/min for the analysis of some nuclear waste samples.
- Table 4 Automated Reagent Delivery Sequence for Am Separation 8
- sample purification apparatus 200 comprises a pump 202 (such as a shown peristaltic pump), a column system 204, and valves 206 and 208.
- Column system 204 can comprise, for example,
- Column system 204 is shown with a single outlet 210 which can, for example, correspond to a single outlet from outlet port 106 of system 80 (Figs. 7 and 8), or can correspond to a joined outlet formed from joining the first and second flow paths of system 50 (Fig. 4) in an embodiment
- Apparatus 200 further comprises a holding coil 212 configured for holding either a sample which is to be separated with column system 204, or a slurry which is to be utilized for forming a packed column.
- Apparatus 200 can have particular application for separating components of biological samples.
- Apparatus 200 permits the sample to be repeatedly cycled across column system 204, which can increase an amount of a biological component ultimately bound to a packed column relative to an amount which would be bound if a sample were not cycled multiple times across
- Strontium was then eluted using a dilute nitric acid solution (ca. 0.05 M).
- the protocol for implementing the separation is shown above in Table 3.
- Reagent solution of 8 M HNO 3 -0.12 M HF was used as a column wash to ensure removal of the tetravalent actinides which were coretained with 90 Sr.
- the renewable column apparatus shown in Figure 8 was applied to separation of 241 Am from nuclear waste sample for determination by alpha- spectroscopy using TRU-resin. Determination of 241 Am in nuclear waste samples using alpha-spectroscopy requires that Am is separated from the stable matrix, highly radioactive fission products, and potential radioactive interferences (e.g., 238 Pu).
- the procedure given in Table 4 was applied to a dissolved vitrified glass nuclear waste sample prepared in 2 M HNO 3 -0.05 M NaNO 2 . The sample was spiked with 5.0 x 10 5 dpm/mL of 241 Am and 39 Pu. On-line radioactivity detection was used to monitor the separation.
- the ammonium bioxalate elution step is required if the TRU-resin column is to be reused for subsequent Am separations.
- the sorbent column can be automatically repacked after the Am elution step. In this case, there was no need to elute the actinides still present on the column (Table 4, step 5). Additional column wash steps to reduce potential carryover into subsequent analysis were also eliminated. The separation time was reduced and mixed waste generation was minimized. This separation approach was applied towards the analysis of nuclear waste samples with off-line detection by alpha spectrometry. Analytical results were in close agreement with those obtained by standard manual protocols.
- This example demonstrates the utility of the renewable microcolumn for DNA purification from complex samples.
- This example includes 60 ⁇ m particles with DNA binding sites, however any DNA binding particles ranging in size from about 10 ⁇ m to 200 ⁇ m could be used with this procedure for DNA purification.
- a schematic of the system used for these experiments is shown in FIG. 10.
- the microcolumn 50 was machined from Plexiglass to include a 1.8 mm diameter column 52.
- the column included a PEEK frit 58 with a lO ⁇ m pore size (Upchurch).
- a syringe pump 122 (Cavro) was used for single pass experiments, and an Eldex reciprocating pump 1002 was added for recirculating the sample through the column 52.
- An eight port valve 1000 was used to direct flow of solutions.
- a typical analytical protocol is outlined in Table E2-1.
- Tepnel microbeads derivatized with universal 1392r oligonucleotide were used for the purification.
- the flowrates for sample injection and DNA elution are average flowrates produced by alternating flow at 3 ⁇ l/s and stopped flow. After the DNA is eluted, the column is automated flushed from the system.
- microbeads for DNA purification were obtained from Tepnel Life Sciences (Cheshire, England) and included universal 16S rDNA oligonucleotide 1392r with a dT8 linker covalently attached to 60 ⁇ m microbeads.
- the binding capacity was estimated to be 2 pmol mg"l (or cm ⁇ ) beads (1.27 x 10 ⁇ 2 capture probes mg ⁇ l) based upon a competitive hot/cold assay using complementary oligonucleotides (Tepnel). Underivatized beads were also obtained to test for non-specific binding of nucleic acids.
- the nucleic acid capture program was initiated by delivering the affinity matrix from a stirred slurry (15 mg ml ⁇ l in 0.3 M NaCl) to the renewable column, resulting in a 7 ⁇ l bed volume (1.6 mm ID x 3.5 mm column height) containing approximately 3.7 mg bead material, 3.5 cm ⁇ surface area and 4.4 x 10*2 capture probes.
- Processing times are summarized for 1 pass of DNA through the renewable column, 10 passes (recirculation) of the DNA sample, and a manual batch reaction using the same reagents. In all cases the sample volume was 200 ⁇ l, and the elution volume was lOO ⁇ l. Processing times in the automated system were not optimized for speed, and therefore do not necessarily reflect a lower limit on processing speed within the system and for other sample matrices.
- Total hybridization time is the sample contact time plus the time elapsed during column washing with the hybridization solution (0.3 M NaCl).
- Nucleic acid extracts (200 ⁇ l) were heat denatured at 100°C for 10 min., quick-chilled on ice and perfused over the microcolumn at 0.9 ⁇ l sec'l for a total contact time of approximately 3 min. The unbound flow-through was collected for subsequent analysis. Beads were washed by perfusing with 80 ⁇ l 0.3M NaCl to remove the nucleic acid extract, and bound nucleic acids were washed in 1.7 ml 0.5X SSC (3 ⁇ l sec"l), and hybridized target was eluted with 100 ⁇ l water at room temperature with a 5.5 min contact time. The total sample processing time from injection to elution was 18 minutes.
- Geobacter 16S rDNA and total eubacterial 16S rDNA were detected and enumerated using dilution-to-extinction PCR to estimate capture efficiency and, for captures in soil extracts, to provide a functional assay for DNA purity that cannot be obtained by scintillation counting of radiolabeled DNA.
- Estimates of capture efficiency were calculated by (PCR detection limit) x (dilution factor) x (conversion factor to account for entire eluant). For example, with a 64 copy detection limit, 53 dilution to the last positive PCR signal, and a 2 ⁇ l DNA input into the 5-fold dilution series (10 % of the total recovered DNA), 8 x 10 ⁇ copies of DNA were recovered.
- the capture efficiency is therefore (8 x 10 ⁇ /1 x l ⁇ 6) x 100, or 8%. Capture efficiencies calculated in this manner are conservative estimates and underestimate the actual capture efficiency. That is, the true extinction point of the PCR lies between the last positive signal and the next dilution in the 5-fold series. Further, the positive control used to calibrate the enumeration was non-fragmented, highly purified DNA rather than sheared DNA isolated from a soil extract (which may amplify with lower efficiency than the standard). These variables bias the calculation of capture efficiency downward, such that the actual capture efficiency calculated in the example above is > 8% but ⁇ 40%. Purified DNA was serial diluted in a 5-fold series immediately prior to
- PCR primers were synthesized by Keystone Labs (Camarillo, CA): Gbc.l300f and Gbc.l400r; S-d401F-20 and S-d683aR-20; universal eubacterial primers fDl and rP2.
- PCR reactions were carried out in 25 ⁇ l total volume, utilizing a Perkin-Elmer (Foster City, CA) 9600 thermal cycler and 0.2 ml thin-walled reaction tubes.
- Final reaction conditions were 2 ⁇ l purified DNA (or dilutions thereof), 10 mM Tris pH 8.3, 50 mM KC1, 2.5 mM MgCl2, 200 ⁇ M each dNTP, 0.2 ⁇ M each primer, and 0.625 U Taq polymerase (Perkin Elmer) which had been pretreated with TaqStartTM antibody at 0.5X the recommended concentration (Sigma, St. Louis, MO).
- Assembled reactions were heated to 80 °C for 5 min (hot start) and amplifications were conducted by performing 5 cycles at 94 °C for 40 s, 60 °C for 10 s, 72 °C for 75 sec followed by 40 cycles at 94°C for 12 s, 65 °C for 10s, 72°C for 80 s with a 2 s extension per cycle. A final 20 min, 72°C extension was performed before chilling reactions to 4°C. Control reactions included no template, solution blank and system washes (pre-and post-capture), affinity-purified nucleic acids (i.e. system eluant) spiked with 250 fg G. chapellii genomic DNA, and a dilution series of G. chapellii genomic DNA.
- PCR conditions for universal eubacterial primers fDl/rP2 were essentially identical, except we utilized 1.5 mM MgCl2 and a thermal profile consisting of 5 cycles at 94°C for 40 s, 55°C for 10 s, 72°C for 75 sec, 30 cycles at 94°C for 12 s, 65°C for 10s, 72°C for 80 s with a 2 s extension per cycle, and a 20 min, 72°C final extension.
- 1 ng of G. chapellii genomic DNA 10 6 cell equivalents (1 fg cell "1 ) or 10 6 copies of target assuming 1 copy per cell.
- the estimated DNA content of the soil extract background was 3 ⁇ g per capture, or ca. 3 x 10 9 competitive DNA targets assuming 5 fg cell " ' and one 16S rDNA target per fg.
- Values in parentheses indicate capture efficiency of total 16S rDNA target based on dilution-to-extinction PCR using universal 16S rDNA primers fdl/rP2.
- the maximal amount of target DNA recovered from 1 ng genomic DNA captures was ca. 80 pg (ca. 8 x 10 ⁇ copies), whereas the amount of target DNA recovered from the 100 ng genomic captures was 6-30 ng (6 x l ⁇ 6 - 3 x 10 ⁇ copies). Therefore, the limited capture of 1 ng target at all size ranges was not due to surface saturation of available binding sites, suggesting that kinetic and/or thermodynamic effects limited nucleic acid capture at the lower target concentrations.
- the capture efficiency for competitive eubacterial 16S rDNA from unspiked soil extract was 0.3%, similar to the capture efficiency of Geobacter genomic DNA targets that were also spiked into the soil background (Table E2-3).
- theG. chapellii 16S rDNA specific capture and total eubacterial 16S rDNA capture were constant and of similar magnitude (0.3% capture efficiency), even though 100 ng of G. chapellii target constituted ca. 3% of the total 16S rDNA and 1 ng represented only 0.03% of total 16S rDNA target.
- the protocol can include passing the sample only one time over the column or recirculating the sample many times over the column.
- the extraction efficiency obtained using the automated system was equal to or better than the manual extraction efficiency using the same reagent.
- DNA (1 ng or lOOng) in a soil extract was detectable by PCR after a single pass over the microcolumn using the automated system and only 18 minutes total processing time. This processing time was not optimized for speed, so does not necessarily reflect a lower limit on processing speed within the system and for other sample matrices. Although the capture efficiency using this reagent was not high, the purified DNA sample does not inhibit PCR detection.
Abstract
Description
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Priority Applications (3)
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EP99925968A EP1082167A1 (en) | 1998-05-27 | 1999-05-26 | Systems for column-based separations, methods of forming packed columns, and methods of purifying sample components |
CA002333371A CA2333371A1 (en) | 1998-05-27 | 1999-05-26 | Systems for column-based separations, methods of forming packed columns, and methods of purifying sample components |
JP2000550578A JP2002516167A (en) | 1998-05-27 | 1999-05-26 | Column-based separation system, packed column formation method, and sample component purification method |
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US8745498A | 1998-05-27 | 1998-05-27 | |
US09/318,345 US6136197A (en) | 1998-05-27 | 1999-05-25 | Systems for column-based separations, methods of forming packed columns, and methods of purifying sample components |
US09/318,345 | 1999-05-25 | ||
US09/087,454 | 1999-05-25 |
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US (4) | US6136197A (en) |
EP (1) | EP1082167A1 (en) |
JP (1) | JP2002516167A (en) |
CA (1) | CA2333371A1 (en) |
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CA2333371A1 (en) | 1999-12-02 |
US20040094482A1 (en) | 2004-05-20 |
EP1082167A1 (en) | 2001-03-14 |
US6136197A (en) | 2000-10-24 |
JP2002516167A (en) | 2002-06-04 |
US6645377B1 (en) | 2003-11-11 |
US6780326B2 (en) | 2004-08-24 |
US20040251206A1 (en) | 2004-12-16 |
US7001522B2 (en) | 2006-02-21 |
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